Nucleic acid hybridization is a known method for identifying specific sequences of nucleic acids. Hybridization is based upon pairing between complementary nucleic acid strands. Single-stranded oligonucleotides having known sequences can be used as probes to identify target sequences of nucleic acid analytes, by exposing the probes to sample solutions containing nucleic acid analytes of interest. If a nucleic acid analyte hybridizes to a probe, the analyte necessarily contains the target sequence. Various aspects of this method have been studied in detail. In essence, all variations allow complementary base sequences to pair and to form double stranded molecules. A number of methods are known in the art to determine whether pairing has occurred, such as those described in U.S. Pat. No. 5,622,822 to Ekeze et al. and U.S. Pat. No. 5,256,535 to Ylikoski et al.
Binding DNA to a substrate coating or substrate may involve chemical moieties that are endogenous or exogenous to the DNA. Generally, exogenous moieties are employed in the end attachment approach. For example, the end attachment approach usually involves providing a binding moiety on either the DNA, the substrate coating surface, or both, with the binding moiety attaching the DNA to the surface. For example, DNA containing an exogenous amine at one terminus can be attached to a surface containing an amine reactive moiety (e.g., an aldehyde, epoxide, isothiocyanate, or isocyanate). See, e.g., U.S. Pat. No. 5,215,882 to Bahl et al., and Guo et al., (1994) Nucleic Acids Research 22: 5456-65. In such a case, a covalent bond is formed between the amine terminated DNA and the amine reactive moiety of the surface. Body attachment is more commonly used for enzymatically prepared probes that may use chemical functionalities endogenous to DNA (See, e.g., Shalon et al. (1996) Genome Research 6,: 639-45) or functionalities that are exogenous to DNA that act as surface binding moieties. Endogenous attachment often involves non-covalent bonding between the surface and the endogenous functionalities. See, e.g., U.S. Pat. No. 5,807,522 to Brown et al. Endogenous attachment techniques have an advantage over exogenous attachment techniques, because there is no need to incorporate additional binding moieties into the DNA, thereby reducing the overall complexity and cost of the process.
When solely endogenous attachment is employed, the surface to which the nucleic acids are to be bound must have reactive sites, e.g., adsorbing or covalent binding moieties, which are capable of covalently or non-covalently binding to endogenous portions of a biomolecule. Examples of such adsorbing or binding moieties can be found in references describing specially modified surfaces for use in solid phase chemistry, including U.S. Pat. Nos. 5,514,785 and 5,667,976 to Van Ness et al., U.S. Pat. Nos. 5,712,383 and 5,747,244 to Sheridan et al., and others. Suitable adsorbing moieties include amide-containing or amine-containing polymers as described in U.S. Pat. Nos. 4,806,631 and 4,806,546 to Carrico et al., PCT Publication No. WO 95/04832, and European Patent Publication No. 458652. Two commonly used attachment surfaces of this type are polylysine-adsorbed glass, and amine-terminated and silated glass.
There are disadvantages, however, to these attachment techniques. It is apparent from the above discussion that exogenous end attachment of polynucleotides requires selective adaption of the polynucleotide termini to bind with the substrate coating surface. Accordingly, the exogenous end attachment technique is not suitable for producing a low cost hybridization assay with a high density of probes bound to a surface. In addition, endogenous body attachment is useful when bound probes are positioned in a manner that allows efficient hybridization to the target. Once the body of a probe nucleotide is endogenously attached to a rigid surface, the mobility of the probe nucleotide is substantially reduced. The significant reduction in mobility compromises the capability of the probe to readily assume a helical conformation and thereby undergo hybridization with the target. Accordingly, endogenous body attachment techniques are inadequate for producing a low-cost hybridization assay with high sensitivity and dynamic range.
Thus, there is a need to provide a method and solid support that allows surface-bound probes sufficient mobility to hybridize efficiently with complementary analytes without exogenous attachment of the probe molecules to the support surface.